Electrophoresis has been developed for the analysis or the separation of substances having an electrical charge, and it is the most powerful means available today particularly for the analysis of proteins. There are acidic, basic and neutral amino acids, and the proteins made of amino acids are classified into acidic, basic and neutral proteins. Proteins in a solution are electrically charged positively or negatively. When the pH of solution of a protein is changed, there is a pH at which the amounts of the positive and negative charges become entirely equal. This pH is called the isoelectric point of the protein. The proteins having an isoelectric point below pH 7 are called acidic proteins, those having an isoelectric point above pH 7 are called basic proteins, and those having an isoelectric point near pH 7 are called neutral proteins. Thus, the electrical charge of a protein is caused by the difference between the isoelectric point and the pH of the solution employed, and proteins can be separated by electrophoresis.
The conventional electrophoresis is divided into free electrophoresis and zone electrophoresis with and without a matrix, respectively. The free electrophoresis is moving boundary electrophoresis where an electric field is directly applied to a sample solution. However, this method is hardly used today because it has numerous defects such as complication of the operation, impossibility of the sample preservation, and disturbance of a pattern due to thermal convection.
The zone electrophoresis is actively used today instead of the free electrophoretic method having the above described defects. A gel-like substance such as agar gel, agarose gel, starch gel and polyacrylamide gel and a porous membrane such as cellulose acetate membrane or filter paper are used as the matrix in the zone electrophoresis and use of these matrices have solved the above described problems of the free electrophoresis.
The characteristic feature of gel electrophoresis which is one of the zone electrophoresis is the possibility of separating proteins by their molecular weights by using the molecular sieving action of the gel-like substance. Thus, when proteins are treated with sodium dodecylsulfate (hereinafter "SDS"), it is possible to unify the amounts of the electrical charges of the proteins, which is one of the two factors to control the electrophoretic mobility of proteins. The electrophoretic mobility depends on the molecular weight of proteins and thus the separation of proteins by molecular weights is possible. Isoelectric focusing which separates the proteins by converging them at their isoelectric points through creation of a pH gradient in this gel is also possible.
The characteristic feature of the porous membrane electrophoresis is its high separability by difference in electrical charge, and its practical simplicity of operation such as requirement of a very minute amount of a sample, extreme shortness of electrophoretic time, and ease of preservation of the electropherogram. Due to these reasons, the porous membrane electrophoresis is suitable for the analysis of proteins in various body fluids such as serum, urine, and spinal fluid. Thus, the gel electrophoresis is suitable for basic research work in medical and biological fields, whereas the porous membrane electrophoresis has been mainly developed as an important diagnostic method in clinical test field.
As described above, the electrical charge and the molecular weight of a protein molecule are important factors which determine the configuration of the protein in a solution. However, hydrophobic bonding is considered to be an even more important factor. Thus, the hydrophobic bonding which functions with water as its medium greatly contributes to the stabilization of proteins. It is considered that higher hydrophobicity of proteins makes the hydrophobic bonding more contribute to the protein structure.
In addition to the classification by electrical charge into acidic, basic and neutral nature as described above, the amino acids constituting proteins are also classified by the properties such as hydrophilicity and hydrophobicity. While alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine and tryptophan which are electrically neutral but have a hydrophobic group in the side chain are typical hydrophobic amino acids, and glycine, serine, threonine, tyrosine, asparagine and glutamine which have a hydrophilic group in the side chain are typical hydrophilic amino acids. Therefore, it is generally considered that hydrophilicity or hydrophobicity of proteins is determined in proportion to the composition of the hydrophilic and hydrophobic amino acids.
As stated above, only the difference in molecular weights and charge density of proteins has been utilized for the separation of proteins in the conventional electrophoresis. The separation utilizing the difference in hydrophilicity and hydrophobicity of proteins, which is another important factor, has never been attempted in conventional electrophoresis. Thus, proteins having similar levels of molecular weights and charge densities but different hydrophilic/hydrophobic properties could not be separated in the past. Therefore, there exists in conventional electrophoresis a serious problem of separating highly hydrophobic lipoproteins or cell membrane associated proteins by the difference in their hydrophobic moieties or of separating highly hydrophobic sugar proteins by the difference in their hydrophilic moieties.
An object of the present invention is to provide a electrophoretic matrix which utilizes the difference in its hydrophobicity or hydrophilicity for the separation of proteins which heretofore could not be separated by prior art electrophoresis.
Another object of this invention is to provide a novel electrophoretic method which employs such an electrophoretic matrix.